1. Field of the Invention
This invention relates to a method to determine the biological function of a target gene in a cell, which comprises: separately culturing a first population and a second population of the cell under same culturing conditions, the first population of the cell differing from the second population of the cell in that the first population of the cell has accepted the introduction of a methylated polynucleotide; comparing the first population and the second population of the cell to determine which biological difference(s) is/are present therebetween; and determining which biological function(s) the target gene is/are associated with based on the determined biological difference(s) between the first population and the second population of the cell.
2. Description of the Related Art
As genomes of numerous species have been sequenced completely, gene function research has drawn attention of scientists. To date, studies aimed at the biological function of a given gene mostly rely on traditional genetic and molecular manipulations such as knock-out, knock-in mutagenesis/transgenic assays, as well as knock down studies using small interfering RNA (siRNA) and antisense RNAs. Amongst these studies, the application of siRNA where the siRNA interferes with the expression of a specific gene has proved to be one of the most powerful means for interrogation of the gene's functions and can be used to target a specific gene or a batch of genes. However, the cost of siRNA is high and siRNA is unstable during delivery. Besides, these studies are all difficult to be expanded from the results of genomic profiling to a high-throughput interrogation of the functions of genes, indicating that their constructions/designs are more time-consuming.
Therefore, there still remains a need in the art to develop a method for functional analysis of specific gene(s) that is less expensive and more stable and convenient in practical use.
In addition to siRNAs, methylated oligonucleotides have been synthesized and used to inhibit the expression of genes as well. For example, U.S. Pat. No. 5,840,497 discloses a method for the silencing of specific genes by DNA methylation. The method involves introducing into a cell a single-stranded oligonucleotide containing 5-methyl deoxycytosine, wherein the single-stranded oligonucleotide has a sequence complementary to a sequence within the promoter region of the gene to be silenced, and wherein the sequence within the promoter region contains at least one CpG doublet.
WO 99/24560 discloses a method of inhibiting the expression of a gene in a cell, comprising the step of administering to the cell a single-stranded oligonucleotide comprising nucleotide units wherein at least one cytosine of a cytosine-guanine base pair contains a methyl group at the 5 position of the cytosine nucleotide.
In this invention, the applicants attempted to develop a method to determine the biological function(s) of a target gene in a cell using a methylated polynucleotide, the sequence of which is identical to or fully complementary to that of a portion of the target gene's nucleotide sequence at the promoter and/or the first exon region thereof. According to the applicants' method, a first population of the cell accepting transfection with the methylated polynucleotide is compared with a second population of the cell without accepting the transfection treatment. The observed biological difference(s) between the first and second populations of the cell is/are then relied upon as a basis for determining the biological function(s) of the target gene in the cell. To prove the practicality and usage of the applicant's method, a gene encoding thyroid hormone receptor interacting protein 10 (TRIP10) was chosen as the target gene and a methylated Trip10 DNA directed to the Trip10 promoter was tested in experiments using either human or rat mesenchymal stem cells (MSCs).
There has been an early report indicating that thyroid hormone receptor interacting protein 10 (TRIP10), also known as Cdc42-interacting protein 4 (CIP4), binds to activated Cdc42 in vitro and in vivo, suggesting that TRIP10 may act as a link between Cdc42 signaling and regulation of the actin cytoskeleton (P. Aspenström (1997), Curr. Biol., 7:479-487). Cdc42 plays a role in cell-cycle control, as it is needed for progression through G1. Recent evidence indicates that Cdc42 has a role in neural progenitors, maintaining them in a self-renewing state, a prerequisite for the maintenance of stem cells into adulthood (S. Cappello et al. (2006), Nature Neuroscience, 9:1099-1107). Thus, TRIP10 association with Cdc42 implies that TRIP10 may be involved in cell-cycle/growth.
It has also been reported that CIP4 accumulation and toxicity in striatal neurons may play a role in Huntington's disease (HD) pathogenesis (S. Holbert et al. (2003), PNAS, 100:2712-2717). Clinical evidence also suggests the interaction of TRIP10 with huntingtin (htt), the protein that regulates both cell growth and apoptosis and its abnormality is identified in the pedigree studies of Huntington's disease (S. Holbert et al. (2003), PNAS, 100:2712-2717). These findings indicate that TRIP10 may be involved in cell growth and apoptosis due to the interaction between TRIP10 and htt. However, the role of the Trip10 gene in cell fate/induction remains largely unknown.
The experimental results obtained by the applicants' method reveal that suppressed Trip10 expression is a critical signal for MSC-to-neuron differentiation and preventing MSCs from death. It is thus believed that the method developed by the applicants is an efficient and powerful tool for analyzing the biological functions of genes.